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The research aims to give new insights on the effect of administering selected bacterial strains, isolated from honey bee gut, and/or a commercial plant extract blend ( HiveAlive®) on Nosema ceranae. Analyses were first performed under laboratory conditions such as different infective doses of N. ceranae, the effect of single strains and their mixture and the influence of pollen administration. Daily survival and feed consumption rate were recorded and pathogen development was analysed using qPCR and microscope counts. Biomarkers of immunity and physiological status were also evaluated for the different treatments tested using one bacterial strain, a mixture of all the bacteria and/or a plant extract blend as treatments. The results showed an increase of abaecin transcript levels in the midgut of the honey bees treated with the bacterial mixture and an increased expression of the protein vitellogenin in the haemolymph of honey bees treated with two separate bacterial strains (Bifidobacterium coryneforme and Apilactobacillus kunkeei). A significant effectiveness in reducing N. ceranae was shown by the bacterial mixture and the plant extract blend regardless of the composition of the diet. This bioactivity was seasonally linked. Quantitative PCR and microscope counts showed the reduction of N. ceranae under different experimental conditions. The antiparasitic efficacy of the treatments at field conditions was studied using a semi-field approach which was adapted from research on insecticides for the first time, to analyse antiparasitic activity against N. ceranae. The approach proved to be reliable and effective in validating data obtained in the laboratory. Both the mixture of beneficial bacteria and its association with Hive Alive ® are effective in controlling the natural infection of N. ceranae in honey bee colonies., peer-reviewed

Background and aims: As potent allelochemicals, flavonoids are believed to be associated with the development of soil-borne diseases. Fusarium species infection is responsible for soybean [Glycine max(L.) Merr.] root rot. However, it is uncertain if and how flavonoids influence rhizosphere Fusarium communities under consecutive soybean monoculture. Methods: Quantitative real-time PCR and pyrosequencing were used to study the Fusarium community. Spectrophotometric techniques and ultra-performance liquid chromatography (UPLC) were used to quantify flavonoids and daidzein and genistein levels, respectively. Results: In field soil, the sizes of Fusarium community were higher from 6 to 13years monoculture than from 1 to 3years. The abundance of Fusarium community was significantly positively correlated with rhizosphere concentrations of the flavonoids and isoflavonoids daidzein and genistein levels. Consecutive monocultured soil selectively inhibited or stimulated certain Fusarium species, F. oxysporum remaining the most abundant. The application of daidzein and a mixture of daidzein and genistein to soil affected the Fusarium community depending on the incubation time and ranging from inhibition to promotion over time. Conclusions: Consecutive soybean monoculture results in shifts in the composition and size of rhizosphere Fusarium community. The Fusarium community was strongly influenced by total flavonoids, in particular daidzein and genistein, rather than soil properties.

Several studies have outlined that changes in the honeybee gut microbial composition may impair important metabolic functions supporting the honeybees’ life. Gut dysbiosis may be caused by diseases like Nosema ceranae or by other anthropic, environmental or experimental stressors. The present work contributes to increasing knowledge on the dynamics of the gut microbiome acquisition in caged honeybees, an experimental condition frequently adopted by researchers, with or without infection with N. ceranae, and fed with a bacterial mixture to control N. ceranae development. Changes of the gut microbiota were elucidated comparing microbial profile of caged and open-field reared honeybees. The absolute abundance of the major gut microbial taxa was studied with both NGS and qPCR approaches, whereas changes in the functionality were based on RAST annotations and manually curated. In general, all caged honeybees showed important changes in the gut microbiota, with $$\gamma$$ γ -proteobacteria (Frischella, Gilliamella and Snodgrassella) lacking in all caged experimental groups. Caged honeybees infected with N. ceranae showed also a strong colonization of environmental taxa like Citrobacter, Cosenzaea and Morganella, as well as possibly pathogenic bacteria such as Serratia. The colonization of Serratia did not occur in presence of the bacterial mixture. The functionality prediction revealed that environmental bacteria or the supplemented bacterial mixture increased the metabolic potential of the honeybee gut microbiome compared to field and caged controls.

The use of secondary treated wastewater (STW) for irrigation is considered as a strategy to mitigate water shortage in summer periods. Unfortunately, the utilization of STW in agriculture is not exempt from environmental and health risks, being a potential source of toxic chemicals (e.g. heavy metals) and human pathogens (e.g. Salmonella spp. and Escherichia coli). The aim of this work is to verify whether irrigation of apple and nectarine trees with STW may lead to heavy metal and microbial contamination in shoot, leaf and fruit tissues. Three-year old trees were grown in pots and drip irrigated separately, for one season, using either tap water (TW) or STW. STW irrigation did not affect heavy metal and trace element concentration (e.g. B, Na, Zn) both in leaves and fruits. Heavy metal concentrations in fruit tissues were lower than international limits for human consumption in both species. Independently from the species, the total bacterial count (TBC) on shoot was almost the double in STW irrigated plants compared to the TW-irrigated ones. No E. coli was found in shoot and fruit; few coliforms (TC) were detected in shoot tissues and in nectarine fruits, far below European microbiological limits for foodstuff. Finally, a laboratory trial, aimed at evaluating possible mechanisms of E. coli translocation inside plants, was carried out on 3-month old peach rootstock plants (i.e. GF 677), artificially inoculated with two E. coli strains, characterized by a different level of pathogenicity (disarmed DH5α and E. coli 1576). Populations of both E. coli strains were isolated as epiphytic and as endophytic only at root level. Nonetheless, the endophytic population of the E. coli 1576 was generally higher than the DH5α strain. Colonization of the epigeal part was never observed. These results are promising for the use of STW, especially for drip irrigated orchards, since water does not wet the canopy and consequently the final product.

The Thematic Group “Plant Health” (TG-PH) of DISTAL, established as part of the “Departments of Excellence” project, has been active since October 2020. To date, 13 research groups with over 40 people including teaching staff, researchers, technicians, postdocs and doctoral students have joined the TG-PH. The common goal of the TG’s activities is to protect and enhance the role of “Plant Health” with a plurality of approaches and specific activities which include: (i) the development of strategies to prevent, monitor and control plant pests; (ii) new agronomic practices and plant nutrition approaches to reduce the impact on ecosystems; (iii) the selection and characterization of new varieties more resilient to biotic/abiotic stresses; (iv) the implementation of sustainable agricultural and food policies. Most of these activities are consistent with initiatives to effectively address major global policy issues such as reducing the use of pesticides and fertilizers, increasing biodiversity in order to ensure healthy, affordable and sustainable food supply to society compliant with food quality and safety. The high interdisciplinarity of the TG-PH is ensured by the wide range of scientific sectors to which the research groups involved belong. The common commitment is to pursue the sustainability objectives of the European Green Deal by bridging the gap between research and practice, accelerating the process of transferring research results and following up with networking. Moreover, TG-PH members share the common belief that only a stronger synergy between this knowledge and expertise coupled with a strong public-private partnership (PPP) will allow for the translation of the results of basic research into concrete solutions useful for the various stakeholders and society at large.

Several studies have outlined that a balanced gut microbiota offers metabolic and protective functions supporting honeybee health and performance. The present work contributes to increasing knowledge on the impact on the honeybee gut microbiota of the three most common veterinary drugs (oxytetracycline, sulfonamides, and tylosin). The study was designed with a semi-field approach in micro-hives containing about 500 honeybees. Micro-hives were located in an incubator during the day and moved outdoors in the late afternoon, considering the restrictions on the use of antibiotics in the open field but allowing a certain freedom to honeybees; 6 replicates were considered for each treatment. The absolute abundance of the major gut microbial taxa in newly eclosed individuals was studied with qPCR and next-generation sequencing. Antimicrobial resistance genes for the target antibiotics were also monitored using a qPCR approach. The results showed that the total amount of gut bacteria was not altered by antibiotic treatment, but qualitative variations were observed. Tylosin treatment determined a significant decrease of α- and β-diversity indices and a strong depletion of the rectum population (lactobacilli and bifidobacteria) while favoring the ileum microorganisms (Gilliamella, Snodgrassella, and Frischella spp.). Major changes were also observed in honeybees treated with sulfonamides, with a decrease in Bartonella and Frischella core taxa and an increase of Bombilactobacillus spp. and Snodgrassella spp. The present study also shows an important effect of tetracycline that is focused on specific taxa with minor impact on alfa and beta diversity. Monitoring of antibiotic resistance genes confirmed that honeybees represent a great reservoir of tetracycline resistance genes. Tetracycline and sulfonamides resistance genes tended to increase in the gut microbiota population upon antibiotic administration. IMPORTANCE This study investigates the impact of the three most widely used antibiotics in the beekeeping sector (oxytetracycline, tylosin, and sulfonamides) on the honeybee gut microbiota and on the spread of antibiotic resistance genes. The research represents an advance to the present literature, considering that the tylosin and sulfonamides effects on the gut microbiota have never been studied. Another original aspect lies in the experimental approach used, as the study looks at the impact of veterinary drugs and feed supplements 24 days after the beginning of the administration, in order to explore perturbations in newly eclosed honeybees, instead of the same treated honeybee generation. Moreover, the study was not performed with cage tests but in micro-hives, thus achieving conditions closer to real hives. The study reaches the conclusion that the most common veterinary drugs determine changes in some core microbiota members and that incidence of resistance genes for tetracycline and sulfonamides increases following antibiotic treatment.

Bees can be severely affected by various plant protection products (PPP). Among these, neonicotinoid insecticides are of concern as they have been shown to be responsible for extensive honeybee colonies death when released into the environment. Also, sublethal neonicotinoid doses contaminating single honeybees and their colonies (e.g. through contaminated pollen) are responsible for honeybees physiological alterations with probable implication also on microbiome functionality. Honeybees show symbiotic interactions with specific gut bacteria that can enhance the adult host performances. Among the known mechanisms, the modulation of the immune system, the degradation of recalcitrant secondary plant metabolites, pollen digestion, and hormonal signaling, are the most important functional benefits for the host honeybee. To date, few research efforts have aimed at revealing the impact of PPP on the gut microbial community of managed and wild honeybees. The majority of the existing literature relays on cage or semifield tests of short duration for research investigating neonicotinoids-gut microbiome interactions. This research wanted to unravel the impact of two neonicotinoids (i.e. imidacloprid and thiacloprid) in natural field conditions up to 5 weeks of exposure. A long-term impact of neonicotinoids on gut microbial community of honeybees was observed. The alterations affected several microbial genera and species such as Frischella spp., lactobacilli and bifidobacteria, whose shifting is implicated in intestinal dysbiosis. Long-term impact leading to dysbiosis was detected in case of exposure to imidacloprid, whereas thiacloprid exposure stimulated temporary dysbiosis. Moreover, the microbial diversity was significantly reduced in neonicotinoid-treated groups. Overall, the reported results support a compromised functionality of the gut microbial community, that might reflect a lower efficiency in the ecosystemic functionality of honeybees.

Social bees harbor a community of gut mutualistic bacteria, among which bifidobacteria occupy an important niche. Recently, four novel species have been isolated from guts of different bumblebees, thus allowing to suppose that a core bifidobacterial population may be present in wild solitary bees. To date there is sparse information about bifidobacteria in solitary bees such as Xylocopa and Osmia spp., this study is therefore focused on the isolation and characterization of bifidobacterial strains from solitary bees, in particular carpenter bee (Xylocopa violacea), builder bee (Osmia cornuta), and red mason bee (Osmia rufa). Among the isolates from Osmia spp. no new species have been detected whereas among Xylocopa isolates four strains (XV2, XV4, XV10, XV16) belonging to putative new species were found. Isolated strains are Gram-positive, lactate- and acetate-producing and possess the fructose-6-phosphate phosphoketolase enzyme. Full genome sequencing and genome annotation were performed for XV2 and XV10. Phylogenetic relationships were determined using partial and complete 16S rRNA sequences and hsp60 restriction analysis that confirmed the belonging of the new strains to Bifidobacterium genus and the relatedness of the strains XV2 and XV10 with XV16 and XV4, respectively. Phenotypic tests were performed for the proposed type strains, reference strains and their closest neighbor in the phylogenetic tree. The results support the proposal of two novel species Bifidobacterium xylocopae sp. nov. whose type strain is XV2 (=DSM 104955T = LMG 30142T), reference strain XV16 and Bifidobacterium aemilianum sp. nov. whose type strain is XV10 (=DSM 104956T = LMG 30143T), reference strain XV4.

In the present work, a combined hurdle approach for fermented meat preservation was investigated. Challenge tests were performed in Chorizo sausage model using the maximum allowed NaNO2 amount (150 mg/kg), a reduced amount (75 mg/kg) and no nitrite, with and without protective cultures inoculation. Cocktail strains of L. monocytogenes and Salmonella spp. were used as indicator strains. In a nitrite reduced sausage model, L. monocytogenes growing trend did not significantly change (p > 0.05) when compared with that containing higher nitrite concentration (150 mg/kg NaNO2). The addition of L. plantarum PSC20 significantly lowered L. monocytogenes growth when compared with control batches without PCS20 (p < 0.05), obtaining 3.84 log cfu/g and 2.62 log cfu/g lower counts in the batches with 150 mg/kg NaNO2 and 75 mg/kg NaNO2 respectively. None of the protective cultures demonstrated in situ antagonistic activity against Salmonella spp. This work pointed out that the reduction of nitrites with the combined use of a protective culture could be a feasible approach to control L. monocytogenes growth in fermented meat foods.

The human intestinal microbiota, establishing a symbiotic relationship with the host, plays a significant role for human health. It is also well known that a disease status is frequently characterized by a dysbiotic condition of the gut microbiota. A probiotic treatment can represent an alternative therapy for enteric disorders and human pathologies not apparently linked to the gastrointestinal tract. Among bifidobacteria, strains of the species Bifidobacterium breve are widely used in paediatrics. B. breve is the dominant species in the gut of breast-fed infants and it has also been isolated from human milk. It has antimicrobial activity against human pathogens, it does not possess transmissible antibiotic resistance traits, it is not cytotoxic and it has immuno-stimulating abilities. This review describes the applications of B. breve strains mainly for the prevention/treatment of paediatric pathologies. The target pathologies range from widespread gut diseases, including diarrhoea and infant colics, to celiac disease, obesity, allergic and neurological disorders. Moreover, B. breve strains are used for the prevention of side infections in preterm newborns and during antibiotic treatments or chemotherapy. With this documentation, we hope to increase knowledge on this species to boost the interest in the emerging discipline known as “therapeutic microbiology„.

The human intestinal microbiota, establishing a symbiotic relationship with the host, plays a significant role for the human health. It is also well known that a disease status is frequently characterized by a dysbiotic condition of the gut. A probiotic treatment can represent an alternative therapy for enteric disorders and human pathologies not apparently linked to the gut environment. Among bifidobacteria, strains of the species Bifidobacterium breve are widely used in pediatrics. B. breve is the dominant species in the gut of breast-fed infants and it has also been isolated from human milk. It has antimicrobial activity against human pathogens, it does not possess transmissible antibiotic resistance traits, it is not cytotoxic and it has immuno-stimulating abilities. This review describes the applications of B. breve strains mainly for the prevention/treatment of pediatric pathologies. The target pathologies range from widespread gut diseases, including diarrhea and infant colics, to celiac disease, obesity, allergic and neurological disorders. Moreover, B. breve strains are used for the prevention of side infections in pre-term newborns and during antibiotic treatments or chemotherapy. With this documentation, we hope to increase knowledge on this species to boost the interest in the emerging discipline known as “therapeutic microbiology”.

Honey bees are important pollinators of several crops and ecosystems, having a great ecological and economic value. In Europe, the restricted use of chemicals and therapeutic agents in the beekeeping sector has stimulated the search for natural alternatives with a special focus on gut symbionts. The modulation of the gut microbiota has been recognised as a practical and successful approach in the entomological field for the management of insect-related problems. To date, only a few studies have investigated the effect of bacterial supplementation on the health status of colonies, colony productivity and gut symbionts. To this purpose, a preparation of sugar syrup containing bifidobacteria and lactobacilli isolated from bee gut was sprayed on the frames of an apiary located in open field once a week for four weeks. Treated and control hives were monitored for two months for brood extension, honey and pollen harvest. The presence of beneficial gut microorganisms within bee gut was investigated with denaturing gradient gel electrophoresis and next generation sequencing. The administered bacteria led to a significant increase of brood population (46.2%), pollen (53.4%) and harvestable honey in honey supers (59.21%). Analysis of the gut microbiota on the new generation of bees in treated hives showed an increase in relative abundance of Acetobacteraceae and Bifidobacterium spp., which are known to be involved in bee nutrition and protection.

Companion animals, also referred as pets, are animals kept primarily as companions at home or having close daily relationship with humans. Dogs and cats are the major human companions and their health and wellness are essential for pet owners. Ensuring a wholesome and balanced diet is a fundamental duty of a responsible pet owner. In the last decades, the growing knowledge in canine and feline nutrition has greatly contributed to improve longevity and well-being of companion animals and interesting outcomes have been obtained with the introduction of dietary fibres and beneficial microorganisms in pets’ diet.

Honeybee is certainly one of the most familiar flying insects of terrestrial habitats. Honeybees are critically important in the environment, sustaining biodiversity and providing essential pollination for a wide range of crops and wild plants. Extensive losses of honeybee colonies in recent years are becoming a major cause of concern. These social insects continuously face threats (diseases, climate change, and management practices) that weaken their health. In this chapter, we will focus on the western honeybee A. mellifera, focusing on gut symbionts, their functions and role on honeybee health

C. jejuni is considered a food safety concern to both public health authorities and consumers since it is the leading bacterial cause of food-borne gastroenteritis in humans. A high incidence of C. jejuni in broiler flocks is often correlated to pathogen recovery in retail poultry meat, which is the main source of human infection. In this work broiler chickens were fed with a synbiotic product mixed with conventional feed using two different administration strategies. The synbiotic was formulated with the microencapsulated probiotic Bifidobacterium longum PCB133 and a xylo-oligosaccharide (XOS). 1-day old chicks were infected with C. jejuni strain M1 (105 cells) and the synbiotic mixture was then administered starting from the first and the 14th day of chicken life (for animal groups GrpC and GrpB respectively). The goal of this study was to monitor C. jejuni load at caecum level at different sampling time by real-time PCR, identifying the best administration strategy. The microbiological analysis of the caecal content also considered the quantification of Campylobacter spp., Bifidobacterium spp. and B. longum. The supplemented synbiotic was more successful in reducing C. jejuni and Campylobacter spp. when administered lifelong, compared to the shorter supplementation (GrpB). Bifidobacterium spp. quantification did not show significant differences among treatments and B. longum PCB133 was detected in both supplemented groups evidencing the successful colonization of the strain. Moreover, the samples of the control group (GrpA) and GrpC were analysed with PCR-denaturing gradient gel electrophoresis (PCR-DGGE) to compare the caecal microbial community profiles at the beginning and at the end of the trial. Pattern analysis evidenced the strong influence of the early synbiotic supplementation, although a physiological change in the microbial community, occurring during growth, could be observed. Experimental results demonstrate that the synbiotic approach at farm level can be an effective strategy, combined with biosecurity measures, to improve the safety of poultry meat.

In order to meet the increasing demand for food quality and safety, the control of pathogenic microorganisms from “farm to fork” is a continuous challenge. This challenge has become more important due to changes in animal production, product processing and distribution, new food habits, higher numbers of consumers at risk for infection and increased awareness. This review is focused on the use of protective and probiotic cultures as “natural” intervention measures to control and prevent the transmission of pathogens along the food chain and on the most used technologies to produce these cultures at the large scale.

Environmental and hereditary factors, together with lifestyle, are important factors in colon cancer development. Considering the increasing incidence of this disease, especially in the developed western world, the last decade has seen much attention directed towards understanding possible prevention strategies. Efforts to study the intestinal microbiota and its interaction with the host have underlined that disbiosis in colonic bacterial composition is a risk factor for colon cancer. Modulation of the composition of intestinal microbiota through the use of probiotic, prebiotic and synbiotic products could therefore represent a strategy for prevention of cancer development. The mechanisms underlying the probiotic-prebiotic anticarcinogenic effect involve a combination of events: e.g. binding of mutagens, suppression of bacteria that convert pro-carcinogens into carcinogens, immune system stimulation, and a reduction in the level of certain intestinal bacterial enzymes that promote carcinogen formation.

Kombucha is usually obtained from the fermentation of black or green tea by a consortium of acetic acid bacteria and yeasts. In this study, kombucha was prepared from the same starter consortium using green and black teas as well as, for the first time, an infusion of rooibos leaves (Aspalathus linearis). Microbial diversity was analysed during fermentation both in the biofilm and in the corresponding kombuchas, using culture-dependent and -independent methods. Polyphenols, flavonoids, ethanol, and acids were quantified and anti-oxidant activities were monitored. All of the Kombuchas showed similarity in bacterial composition, with the dominance of Komagataeibacter spp. Beta diversity showed that the yeast community was significantly different among all tea substrates, between 7 and 14 days of fermentation and between biofilm and kombucha, indicating the influence of the substrate on the fermenting microbiota. Kombucha from rooibos has a low ethanol concentration (1.1 mg/mL), and a glucuronic acid amount that was comparable to black tea. Although antioxidant activity was higher in black and green kombucha compared to rooibos, the latter showed an important effect on the recovery of oxidative damage on fibroblast cell lines against oxidative stress. These results make rooibos leaves interesting for the preparation of a fermented beverage with health benefits.

Nowadays, honey bees are stressed by a number of biotic and abiotic factors which may compromise to some extent the pollination service and the hive productivity. The EU ban of antibiotics as therapeutic agents against bee pathogens has stimulated the search for natural alternatives. The increasing knowledge on the composition and functions of the bee gut microbiota and the link between a balanced gut microbiota and health status have encouraged the research on the use of gut microorganisms to improve bee health. Somehow, we are assisting to the transfer of the “probiotic concept” into the bee science. In this review, we examine the role of the honey bee gut microbiota in bee health and critically describe the available applications of beneficial microorganisms as pest control agents and health support. Most of the strains, mainly belonging to the genera Lactobacillus, Bifidobacterium and Bacillus, are isolated from honey bee crop or gut, but some applications involve environmental strains or formulation for animal and human consumption. Overall, the obtained results show the favourable effect of applied microbial strains on bee health and productivity, in particular if strains of bee origin are used. However, it is actually not yet possible to conclude whether this strategy will ever work. In particular, many aspects regarding the overall setup of the experiments, the dose, the timing and the duration of the treatment need to be optimized, also considering the microbiological safety of the hive products (i.e. pollen and honey). In addition, a deep investigation about the effect on host immunity and physiology is envisaged. Lastly, the final users of the formulations, i.e. beekeepers, should be taken into account for the achievement of high-quality, cost-effective and easy-to-use products.

Nosema ceranae is a widespread microsporidium of European honeybee Apis mellifera L. affecting bee health. The ban of Fumagillin-B (dicyclohexylammonium salt) in the European Union has driven the search for sustainable strategies to prevent and control the infection. The gut microbial symbionts, associated to the intestinal system of vertebrates and invertebrates and its impact on host health, are receiving increasing attention. In particular, bifidobacteria and lactobacilli, which are normal inhabitants of the digestive system of bees, are known to protect their hosts via antimicrobial metabolites, immunomodulation and competition. In this work, the dietary supplementation of gut bacteria was evaluated under laboratory conditions in bees artificially infected with the parasite and bees not artificially infected but evidencing a low natural infection. Supplemented bacteria were selected among bifidobacteria, previously isolated, and lactobacilli, isolated in this work from healthy honeybee gut. Four treatments were compared: bees fed with sugar syrup (CTR); bees fed with sugar syrup containing bifidobacteria and lactobacilli (PRO); bees infected with N. ceranae spores and fed with sugar syrup (NOS); bees infected with N. ceranae and fed with sugar syrup containing bifidobacteria and lactobacilli (NP). The sugar syrup, with or without microorganisms, was administered to bees from the first day of life for 13 days. N. ceranae infection was carried out individually on anesthetised 5-day-old bees. Eight days after infection, a significant (P

Background and aims: Endophytic bacteria have been often studied as biological control agents of plant pathogens and many of their secondary metabolites involved in antagonism are Non Ribosomal Peptides (NRPs). In this study, the molecular basis of the biocontrol properties of the endophyte Pseudomonas synxantha, isolated from Actinidia chinensis, against Pseudomonas syringae pv. actinidiae (Psa), the causal agent of bacterial kiwi canker, has been investigated. Methods: Antagonism-deficient mutants of P. synxantha strain DLS65 were generated by insertion of minitransposon mTn5-GNm in its genome. Southern blot analysis allowed the selection of single transposon insertion-mutants. Amplification of the transposon flanking regions by means of arbitrary and single primer PCR in selected mutants was perfomed to obtain amplicons for sequencing purposes. Results: Sequencing results of the amplicons obtained from three antagonism-deficient mutants led to the localization of the transposonin three genes, which implies their involvement in the antagonism of P. synxantha: an acyl-homoserine lactone acylase gene (pvdQ), a glucose-6-phosphate dehydrogenase gene (zwf) and an mbtH-like gene were identified. It is known from the literature that these three genes are involved directly or indirectly in NRPs synthesis. Conclusions: We suggest that a molecule with antibiotic properties, produced by NRP synthetases, contributes to the antagonistic activity of P. synxantha.

A PCR-RFLP technique has been applied on 13 species of Bifidobacterium in order to update a previous study carried out by Baffoni et al. [1]. This method is based on the restriction endonuclease activity of HaeIII on the PCR-amplified hsp60 partial gene sequence, and allows a rapid and efficient identification of Bifidobacterium spp. strains at species and subspecies level.

Background: Fusarium head blight (FHB) is a severe disease caused by different Fusarium species, which affects a wide range of cereal crops, including wheat. It determines from 10 to 30 % of yield loss in Europe. Chemical fungicides are mainly used to reduce the incidence of FHB, but low environmental impact solutions are looked forward. Applications of soil/rhizobacteria as biocontrol agents against FHB in wheat are described in literature, whereas the potential use of lactobacilli in agriculture has scarcely been explored. Results: The aim of this work was to study the inhibitory effect of two bacterial strains, Lactobacillus plantarum SLG17 and Bacillus amyloliquefaciens FLN13, against Fusarium spp. in vitro and to assess their efficacy in field, coupled to the study of the microbial community profile of wheat seeds. Antimicrobial assays were performed on agar plates and showed that the two antagonistic strains possessed antimicrobial activity against Fusarium spp. In the field study, a mixture of the two strains was applied to durum wheat i) weekly from heading until anthesis and ii) at flowering, compared to untreated and fungicide treated plots. The FHB index, combining both disease incidence and disease severity, was used to evaluate the extent of the disease on wheat. A mixture of the two microorganisms, when applied in field from heading until anthesis, was capable of reducing the FHB index. Microbial community profile of seeds was studied via PCR-DGGE, showing the presence of L. plantarum SLG17 in wheat seeds and thus underlining an endophytic behavior of the strain. Conclusions: L. plantarum SLG17 and B. amyloliquefaciens FLN13, applied as biocontrol agents starting from the heading period until anthesis of wheat plants, are promising agents for the reduction of FHB index.

The study aimed to investigate the antibacterial activity of Melia azedarach fruit extracts in plant shoot culture. Unripe fruits were collected in different periods and the extracts were obtained through homogenization in an ethanol (EtOH) solution (40 g fresh weight in 100 ml 50 % EtOH) followed by EtOH evaporation. Their activity was tested against isolates of several bacterial species (Bacillus spp., Pseudomonas spp.) of agronomic interest, and on shoots of ‘MRS 2/5’ (Prunus cerasifera x Prunus spinosa) and calla lily (Zantedeschia aethiopica) inoculated (bacterized) with Microbacterium oleivorans (Mo), Bacillus subtilis (A17), Sphingomonas meloni (Sm) and Bacillus nealsonii (Bn). Shoot number and fresh weight of explants of ‘MRS 2/5’ on the proliferation medium and shoot length on the rooting medium were reduced in cultures derived from shoots bacterized with Mo and A17. Moreover, rooting frequency and root number decreased in A17-bacterized shoots. In calla lily, the number of rooted shoots was strongly reduced also by Sm. The extracts had a bactericidal activity against Mo, A17, Bn and most other Bacillus spp. isolates within 2-year trials. This activity remained unchanged against most of the tested bacterial strains after autoclave-sterilization (20 min at 120 °C). The filter-sterilized (0.22-μm Millipore membrane) extract generally reduced shoot elongation, and caused the death of some ‘MRS 2/5’shoots at a 20 % level in the proliferation medium, while all calla lily shoots survived the treatment. It strongly reduced Mo and Bn spread in both plant genotypes, and was even bactericidal against Mo in’MRS 2/5’, without showing any negative effect on growth of survived shoots already at the second subculture on standard medium. The HPTLC analysis revealed the prevalence of polyphenols in the extracts from fruits collected in two successive periods; this was consistent with their antimicrobial activity.

The extensive use of silver nanoparticles (SNPs) as antimicrobial in food, clothing and medicine, leads inevitably to a loss of such nanomaterial in soil and water. Little is known about the effects of soil contamination, in particular, on microbial cells, which play a fundamental ecological role. In this work, the impact of SNPs on forest soil has been studied, investigating eco-physiological indicators of microbial biomass and microbial diversity with culture-dependent and independent techniques. Moreover, SNPs bioavailability and uptake were assessed. Soil samples were spiked with SNPs at two different concentrations (10 and 100 μg g(-1)dw) and incubated with the relative controls for 30, 60 and 90 days. The overall parameters showed a significant influence of the SNPs on the soil microbial community, revealing a marked shift after 60 days of incubation.

Normalization is an essential prerequisite for producing accurate real-time PCR expression analyses. The objective of this study is the selection of a set of optimal reference genes in Bifidobacterium adolescentis gene expression studies under bile exposure. B adolescentis is a particularly abundant species in the human adults gut microbiota, exerting relevant probiotic activities. In the gastrointestinal tract, bile represents a hard challenge for bacterial survival, because of its toxic effect. The natural exposure to bile in the colonic environment induces cells adaptation and tolerance mechanisms in bifidobacteria, which determines changes in gene expression profile, influencing the expression levels of housekeeping genes. In this context, the stability of 9 putative reference genes (cysS, purB, recA, rpoB-L, GADPH-R, 16S rRNA, glnA1, gyrA2, sdhA) was examined in B. adolescentis exposed to bile extract, using two different software (BestKeeper and NormFinder). Both algorithms identified gyrA2 and sdhA as the most stable genes under our experimental conditions, while 16S rRNA is the least reliable HKGs. To our best knowledge, this is the first attempt to validate reference genes in Bifidobacterium spp. and the results offer an appropriate set of reference genes suitable for qRT-PCR studies on B. adolescentis strains under bile stress.

Turfgrasses are not only designed for recreation activities, but they also provide beneficial environmental effects and positively influence the human wellness. Their major problems are predisposition to tearing out and microbial diseases. The aim of this study was to investigate whether the inoculation of microorganisms can be effective to improve plant growth and root development of perennial ryegrass, to evaluate new sustainable practice for green preservation. A microorganism-based commercial product was used to amend hydroponically grown Lolium perenne L. and results compared with the use of the same filtered product, a phytohormone solution and an untreated control. Plants were grown for five weeks, shoots cut and measured at one-week interval and, at the end, roots were measured for length and weight. Shoot resistance to tearing out was also tested. Moreover, the main microbial groups present in the product were characterized and the microbial profile of sand and root samples was investigated by PCR-DGGE. The plants treated with the product showed an increased resistance to tearing out with respect to other treatments and roots were longer with respect to the control. Microbial analyses of the product evidenced bacterial and yeast species with plant growth promoting activity, such as Stenothrophomonas maltophilia, Candida utilis and several Lactobacillus species. Some Lactobacillus strains were also found to be able to colonize plant roots. In conclusion, the treatment with microorganisms has a great potential for the maintenance and increased performance of turfgrass surfaces.

With the ban of dietary antimicrobial agents, the use of probiotics, prebiotics and synbiotics has attracted a great deal of attention in order to improve intestinal health and control food-borne pathogens, which is an important concern for the production of safe meat and meat products. Recently, Campylobacter jejuni has emerged as a leading bacterial cause of food-borne gastroenteritis in humans, and epidemiological evidences indicate poultry and poultry products as the main source of human infection. This work aimed at the development of a synbiotic mixture capable of modulating the gut microbiota of broiler chickens to obtain an increase of the beneficial bacteria (i.e. bifidobacteria, lactobacilli) and a competitive reduction of C. jejuni. The prebiotic compound used in the mixture was chosen after an in vivo trial: a fructooligosaccharide and a galactooligosaccharide were separately administered to broilers mixed with normal feed at a concentration of 0.5% and 3%, respectively. Quantitative PCR on DNA extracted from fecal samples revealed a significant (p

Campylobacteriosis is at present the most frequent zoonosis in humans and the main source is poultry meat contaminated by Campylobacter jejuni. An alternative and effective approach to antibiotic administration to livestock to reduce bacterial contamination is the use of probiotics, which can help to improve the natural defence of animals against pathogenic bacteria. In this study 55 lactic acid bacteria and bifidobacteria were screened for desirable properties for their application as probiotics against Campylobacter in poultry. All bacteria were examined for their antimicrobial activity against three C. jejuni strains. Strains exhibiting the highest anti-Campylobacter activity were examined for their survival in the gastro intestinal tract (low pH and presence of bile salts) and food/feed processing conditions (high temperature, high NaCl concentration and starvation) and basic safety aspects such as antibiotic susceptibility and hemolytic activity were studied. On the basis of these activities, two strains, namely Lactobacillus plantarum PCS 20 and Bifidobacterium longum PCB 133, were chosen for an in vivo trial in poultry. They were separately administered to healthy chickens in order to evaluate their capability of colonizing the GI tract of poultry and to estimate their effect on C. jejuni population. The results evidenced that L. plantarum PCS 20 was not present in poultry feces at detectable concentration, whereas B. longum PCB 133 significantly increased after two weeks of daily administration and its amount was still high after a wash-out period of 6 days. In the same period, C. jejuni concentration in poultry feces was significantly reduced in chickens administered with B. longum PCB 133. Therefore, B. longum PCB 133, possessing interesting probiotic properties and a marked anti-Campylobacter activity both in vitro and in vivo, is an excellent candidate for being employed as additives to feed for poultry for the reduction of food-borne campylobacteriosis in humans.

BACKGROUND: Bifidobacterium represents one of the largest genus within the Actinobacteria, and includes at present 32 species. These species share a high sequence homology of 16S rDNA and several molecular techniques already applied to discriminate among them give ambiguous results.The slightly higher variability of the hsp60 gene sequences with respect to the 16S rRNA sequences offers better opportunities to design or develop molecular assays, allowing identification and differentiation of closely related species. hsp60 can be considered an excellent additional marker for inferring the taxonomy of the members of Bifidobacterium genus. RESULTS: This work illustrates a simple and cheap molecular tool for the identification of Bifidobacterium species. The hsp60 universal primers were used in a simple PCR procedure for the direct amplification of 590 bp of the hsp60 sequence. The in silico restriction analysis of bifidobacterial hsp60 partial sequences allowed the identification of a single endonuclease (HaeIII) able to provide different PCR-restriction fragment length polymorphism (RFLP) patterns in the Bifidobacterium spp. type strains evaluated. The electrophoretic analyses allowed to confirm the different RFLP patterns. CONCLUSIONS: The developed PCR-RFLP technique resulted in efficient discrimination of the tested species and subspecies and allowed the construction of a dichotomous key in order to differentiate the most widely distributed Bifidobacterium species as well as the subspecies belonging to B. pseudolongum and B. animalis.

Culture-dependent and culture-independent techniques were applied to investigate the bacterial communities occurring in 5-6 days-old honey bee larvae (Apis mellifera L.), with evident symptoms of disease and healthy-looking ones. Samples were collected by the Bee Emergency Service Team (BeeNet Project) from two apiaries located in different geographical sites (North and South Italy). Observed symptoms were atypical, but very close to those attributed to the European foulbrood (EFB) and to the American foulbrood (AFB), which are severe diseases affecting honey bee larvae worldwide. Isolates from diseased larvae were identified as Enterococcus faecalis Schleifer et Kilpper-Balz and Paenibacillus dendritiformis Tcherpakov et al.. Melissococcus plutonius (ex White) Bailey et Collins corrig. Truper et de Clari, the causative agent of EFB, was detected by polymerase chain reaction in both diseased and healthy-looking larval samples, whereas Paenibacillus larvae White, the causative agent of the AFB, failed to be detected. Microbial profiles obtained by denaturing gradient gel electrophoresis did not show relevant differences among samples, thus evidencing that the healthy-looking samples were partially affected. Besides confirming the presence of E. faecalis and P. dendritiformis, as found by plate count, the technique confirmed the presence of M. plutonius in all samples. The study has evidenced that honey bee larvae were affected by the EFB, with the presence of an atypical Paenibacillus species as second invader, which presumably confers a different symptomatology to the diseased brood.

L’incidenza delle malattie trasmesse da alimenti di origine animale è in costante ascesa in tutti i Paesi industrializzati; si crea, perciò, la necessità di attuare misure di controllo sempre più restrittive negli allevamenti, soprattutto a seguito del divieto sull’uso degli antibiotici come promotori della crescita a scopo sub-terapeutico. I principali patogeni che si ritrovano nei prodotti di origine animale, a volte veicolati nel tratto gastrointestinale degli animali in maniera asintomatica, sono: Campylobacter jejuni, Clostridium perfringens, E. coli 0157:H7, Listeria monocytogenes, Salmonella, Staphylococcus aureus. È necessario trovare, velocemente, soluzioni alternative per controllare e prevenire la diffusione dei patogeni enterici, al fine di ridurre la loro presenza nel prodotto finito e limitare la minaccia per la salute dell’uomo. La modulazione del tratto gastrointestinale, attraverso l’utilizzo di additivi alimentari come probiotici e prebiotici, offre una valida soluzione per mantenere gli animali in salute, ridurre il rischio di contrarre malattie gastrointestinali e migliorare la qualità delle carni destinate al consumo umano. In questo articolo verranno discusse le più recenti applicazioni di probiotici e prebiotici in campo animale, volte alla riduzione della presenza di microrganismi patogeni, potenziali cause di intossicazioni alimentari, nelle carni.